The long-term goal of the proposed work is to elucidate the process leading to programmed cell death (apoptosis) by applying a new and general method we have developed for global proteomic profiling of proteolysis ("degradomics"). Our hypothesis is that proteases, and in particular caspases, act like demolition experts to target key control points to disrupt cellular homeostasis. Finding these points of attack, identifying the proteases involved, and determining the sequence of events are all fundamental to understanding cell death. Apoptosis is a key cellular process that regulates diseases such as cancer, stroke and neurodegenerative diseases among others. By discovering the critical regulatory points in apoptosis, new and important drug targets for these diseases may be identified. Apoptosis is dominated by proteolytic events that are primarily mediated by caspases, aspartate specific proteases. However, the protein substrates they cleave and the cascades they ignite are only partially understood. This proposal applies a new and general method that allows one to selectively label and enrich for a-amino peptides, the product of proteolysis. Tagged products can be readily identified and sequenced by LC/MS/MS. Preliminary studies in Jurkat cells induced to apoptose have already identified 68 asparate cleavage sites of which two-thirds are new substrates. Data suggest the total set of caspase substrates may be up to 3 to 6-times larger than the entire data base of reported caspase substrates across all cells. A combination of cellular ("forward") and in vitro ("reverse") degradomics experiments are proposed. These experiments will provide the most comprehensive map to date of proteins cleaved and proteases responsible for intrinsic and extrinsic apoptosis pathways. There are over a 1000 different human protease genes. The degradomics approaches developed here for caspases will be general and applicable to mapping other protease signaling events. Proteases are involved in most areas of biology and mediate many diseases. It is known that prosurvival proteins are cleaved during apoptosis and many cancer treatments involve compounds that selectively induce apoptosis through inhibiting prosurvival proteins in cancer cells. Thus, in addition to mapping the process of apoptosis and demonstrating a general degradomics method, the proposed work may identify new drug targets for the development of cancer therapies.